RESUMEN
Natural Killer (NK) cells perform many functions that depend on actin assembly, including adhesion, chemotaxis, lytic synapse assembly and cytolysis. HS1, the hematopoietic homolog of cortactin, binds to Arp2/3 complex and promotes actin assembly by helping to form and stabilize actin filament branches. We investigated the role of HS1 in transendothelial migration (TEM) by NK cells. Depletion of HS1 led to a decrease in the efficiency of TEM by NK cells, as measured by transwell assays with endothelial cell monolayers on porous filters. Transwell assays involve chemotaxis of NK cells across the filter, so to examine TEM more specifically, we imaged live-cell preparations and antibody-stained fixed preparations, with and without the chemoattractant SDF-1α. We found small to moderate effects of HS1 depletion on TEM, including whether the NK cells migrated via the transcellular or paracellular route. Expression of HS1 mutants indicated that phosphorylation of HS1 tyrosines at positions 222, 378 and 397 was required for rescue in the transwell assay, but HS1 mutations affecting interaction with Arp2/3 complex or SH3-domain ligands had no effect. The GEF Vav1, a ligand of HS1 phosphotyrosine, influenced NK cell transendothelial migration. HS1 and Vav1 also affected the speed of NK cells migrating across the surface of the endothelium. We conclude that HS1 has a role in transendothelial migration of NK cells and that HS1 tyrosine phosphorylation may signal through Vav1.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Cortactina/metabolismo , Células Asesinas Naturales/metabolismo , Migración Transendotelial y Transepitelial , Proteínas Adaptadoras Transductoras de Señales , Proteínas Sanguíneas/genética , Línea Celular , Cortactina/genética , Humanos , Células Asesinas Naturales/fisiología , Mutación , Fosforilación , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismoRESUMEN
Endothelial cells (ECs) line the microvasculature and constitute a barrier between the vessel lumen and surrounding tissues. ECs inform circulating immune cells of the health and integrity of surrounding tissues, recruiting them in response to pathogens and tissue damage. ECs play an active role in the transmigration of immune cells across the vessel wall. We have discovered important differences in the properties of ECs on soft hydrogel substrates of varying stiffness, in comparison to glass. Primary ECs from several human sources were tested; all formed monolayers normally on soft substrates. EC monolayers formed more mature cell-cell junctions on soft substrates, relative to glass, based on increased recruitment of vinculin and F-actin. EC monolayers supported transendothelial migration (TEM) on soft substrates. Immune cells, including peripheral blood lymphocytes (PBLs) and natural killer cells, showed decreasing numbers of paracellular (between-cell) transmigration events with decreasing substrate stiffness, while the number of transcellular (through-cell) events increased for PBLs. Melanoma cancer cells showed increased transmigration with decreased stiffness. Our findings demonstrate that endothelial monolayers respond to the mechanical properties of their surroundings, which can regulate the integrity and function of the monolayer independently from inflammatory signals. Soft hydrogel substrates are a more appropriate and physiological model for tissue environments than hard substrates, with important implications for the experimental analysis of TEM.
